Project Summary/Abstract Faithful replication of the genetic materials is critical for normal embryonic development, cellular differentiation and reprogramming. Alterations in either gene copy number, structure of one or more chromosomes, or the number of whole chromosomes result in aneuploidy or chromosome somatic copy number alterations/SCNAs. Aneuploidy has been found in 90% of the human solid tumors and is linked to poor prognosis. On the other hand, aneuploidy has been documented to be an integral part of differentiation of mouse trophoblast giant cells (TGC), cells analogous to invasive extravillous trophoblast (EVT) in the human placenta. Both TGC and EVT are involved in remodeling of maternal vessels and establishment of the maternal-fetal interface during implantation. These processes involve interaction between TGC/EVT and the maternal immune system, during which the placental cells escape apoptosis, similar to cancer cells. Abnormal differentiation or dysfunction of TGC/EVT leads to severe developmental consequences, including impaired placental development and fetal growth restriction. This project aims to substantially improve our knowledge of the cellular differentiation of human EVTs regarding their genomic landscape and their interaction with the maternal immune system. Building on our preliminary data, we propose that similar to cancer cells, the development of aneuploidy/SCNAs in TGC/EVTs is associated with cellular mechanisms which promote interaction with the maternal immune system. Specifically, we will first aim to use a genomics approach to interrogate the nature of aneuploidy/SCNA in primary human EVT. We will perform whole genome sequencing (WGS) on isolated EGFR+ cytotrophoblast (CTB) and HLAG+ EVT from normal term placentas as well as CTB and in vitro-differentiated EVTs from first trimester placentas. We expect to obtain a comprehensive landscape of the trophoblast genome. Because one important consequence of aneuploidy is induction of proteotoxic stress or activation of ER stress/unfolded protein response (UPR) pathway, we hypothesize that aneuploidy leads to proteotoxic stress in EVT, inducing ER stress/UPR pathway and leading to externalization of calreticulin, and that this process is required for interactions between EVT and maternal macrophages. We have identified calreticulin and ER stress/UPR as a gene or pathway enriched in EVT. In addition, our preliminary immunohistochemical staining studies show calreticulin to be enriched in EVT. We will determine the role of ER stress/UPR pathway and calreticulin in EVT differentiation, using a combination of lentiviral based gene knockdown and inhibitors. We will determine whether the outcome of the immune synapse between EVT and maternal macrophages is dependent on externalization of calreticulin. The successful completion of this project will significantly advance the field of human trophoblast biology, by demonstrating for the first time that regulated genetic alterations are functionally linked to the normal differentiation of trophoblast, enabling proper placentation through interaction with the maternal immune system. !